U.S. patent application number 10/317853 was filed with the patent office on 2004-01-15 for real time detection of antigens.
This patent application is currently assigned to Utah State University. Invention is credited to Walsh, Marie K., Weimer, Bart C..
Application Number | 20040009529 10/317853 |
Document ID | / |
Family ID | 30118991 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009529 |
Kind Code |
A1 |
Weimer, Bart C. ; et
al. |
January 15, 2004 |
Real time detection of antigens
Abstract
Antigens can be captured and detected from complex samples, such
as food and environmental samples, in about 30 minutes using
apparatus and methods that include flow of the samples through a
module containing antibodies coupled to beads. The samples flow
through the modified beads at about 0.2 to 1.2 liters/minute, which
fluidizes the bead bed. The antigens are captured by the
antibodies, and then detection of the captured antibodies is
carried out with chemiluminescence, fluorescence, or
spectrophotometric techniques.
Inventors: |
Weimer, Bart C.; (Logan,
UT) ; Walsh, Marie K.; (North Logan, UT) |
Correspondence
Address: |
ALAN J. HOWARTH
P.O. BOX 1909
SANDY
UT
84091-1909
US
|
Assignee: |
Utah State University
|
Family ID: |
30118991 |
Appl. No.: |
10/317853 |
Filed: |
December 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10317853 |
Dec 11, 2002 |
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10163253 |
Jun 4, 2002 |
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10163253 |
Jun 4, 2002 |
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09292172 |
Apr 15, 1999 |
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6399317 |
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60081889 |
Apr 15, 1998 |
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Current U.S.
Class: |
435/7.1 ;
436/523 |
Current CPC
Class: |
G01N 33/54353 20130101;
G01N 33/54306 20130101; G01N 33/54366 20130101; G01N 33/56911
20130101; G01N 33/5304 20130101 |
Class at
Publication: |
435/7.1 ;
436/523 |
International
Class: |
G01N 033/53; G01N
033/543 |
Claims
The subject matter claimed is:
1. A method for capturing and concentrating a selected antigen from
an aqueous medium containing a mixture of antigens comprising: (a)
causing a first volume of the aqueous medium containing the mixture
of antigens to flow through a module containing at least two
antibody-bead conjugates, wherein each of the antibody-bead
conjugates comprises a bead, a polymeric spacer covalently coupled
to the bead, and an antibody covalently coupled to the polymeric
spacer, wherein the antibody is configured for binding the selected
antigen, at a first flow rate such that the antibody-bead
conjugates form a fluidized bed and the selected antigen binds to
the antibody-bead conjugates; (b) washing the antibody-bead
conjugates having the selected antigen bound thereto by causing a
wash medium to flow through the module at a second flow rate such
that the antibody-bead conjugates having the selected antigen bound
thereto form a fluidized bed; and (c) holding the washed
antibody-bead conjugates having the selected antigen bound thereto
in a second volume of a second wash medium, wherein the second
volume is smaller than the first volume.
2. The method of claim 1 wherein the aqueous medium containing the
mixture of antigens comprises a food.
3. The method of claim 1 wherein the aqueous medium containing the
mixture of antigens comprises an environmental sample.
4. The method of claim 1 wherein the bead is a glass bead.
5. The method of claim 1 wherein the bead is a ceramic bead.
6. The method of claim 1 wherein the bead has a diameter of about 1
to 7 millimeters.
7. The method of claim 1 wherein the polymeric spacer comprises
dextran.
8. The method of claim 1 wherein the polymeric spacer comprises
polyethylene glycol.
9. The method of claim 1 wherein the polymeric spacer comprises a
polyamino acid.
10. The method of claim 9 wherein the polyamino acid comprises
polythreonine.
11. The method of claim 9 wherein the polyamino acid comprises
polyserine.
12. The method of claim 1 wherein the antibody comprises a
monoclonal antibody.
13. The method of claim 1 wherein the antibody comprises a
polyclonal antibody.
14. The method of claim 1 wherein the antibody comprises an
antibody fragment.
15. The method of claim 1 wherein flow is caused by pumping.
16. The method of claim 1 wherein flow is caused by applying
partial vacuum.
17. The method of claim 1 wherein said first flow rate is about 0.2
to 1.2 liters/minute.
18. The method of claim 17 wherein the first flow rate is about 0.3
to 0.7 liters per minute.
19. A method for capturing and concentrating a selected antigen
from an aqueous medium containing a mixture of antigens comprising:
(a) causing a first volume of the aqueous medium containing the
mixture of antigens to flow through a module containing at least
two antibody-bead conjugates, wherein each of the antibody-bead
conjugates comprises a 1-7 millimeter glass or ceramic bead, a
dextran or polyethylene glycol spacer covalently coupled to the
bead, and an antibody covalently coupled to the spacer, wherein the
antibody is configured for binding the selected antigen, at a first
flow rate such that the antibody-bead conjugates form a fluidized
bed and the selected antigen binds to the antibody-bead conjugates;
(b) washing the antibody-bead conjugates having the selected
antigen bound thereto by causing a wash medium to flow through the
module at a second flow rate such that the antibody-bead conjugates
having the selected antigen bound thereto form a fluidized bed; and
(c) holding the washed antibody-bead conjugates having the selected
antigen bound thereto in a second volume of a second wash medium,
wherein the second volume is smaller than the first volume.
20. A method for detecting a selected antigen in aqueous medium
containing a mixture of antigens comprising: (a) causing the
aqueous medium containing the mixture of antigens to flow through a
module containing at least two antibody-bead conjugates, wherein
each of the antibody-bead conjugates comprises a bead, a polymeric
spacer covalently coupled to the bead, and an antibody covalently
coupled to the polymeric spacer, wherein the antibody is configured
for binding the selected antigen, at a first flow rate such that
the antibody-bead conjugates form a fluidized bed and the selected
antigen binds to the antibody-bead conjugates; (b) washing the
antibody-bead conjugates having the selected antigen bound thereto
by causing a first wash medium to flow through the module at a
second flow rate such that the antibody-bead conjugates having the
selected antigen bound thereto form a fluidized bed; and (c)
detecting the selected antigen bound to the antibody-bead
conjugates by enzyme-linked immunosorbent assay.
21. The method of claim 20 wherein the aqueous medium containing
the mixture of antigens comprises a food.
22. The method of claim 20 wherein the aqueous medium containing
the mixture of antigens comprises an environmental sample.
23. The method of claim 20 wherein the bead is a glass bead.
24. The method of claim 20 wherein the bead is a ceramic bead.
25. The method of claim 20 wherein the bead has a diameter of about
1 to 7 millimeters.
26. The method of claim 20 wherein the polymeric spacer comprises
dextran.
27. The method of claim 20 wherein the polymeric spacer comprises
polyethylene glycol.
28. The method of claim 20 wherein the polymeric spacer comprises a
polyamino acid.
29. The method of claim 28 wherein the polyamino acid comprises
polythreonine.
30. The method of claim 28 wherein the polyamino acid comprises
polyserine.
31. The method of claim 20 wherein the antibody comprises a
monoclonal antibody.
32. The method of claim 20 wherein the antibody comprises a
polyclonal antibody.
33. The method of claim 20 wherein the antibody comprises an
antibody fragment.
34. The method of claim 20 wherein flow is caused by pumping.
35. The method of claim 20 wherein flow is caused by applying
partial vacuum.
36. The method of claim 20 wherein said first flow rate is about
0.2 to 1.2 liters/minute.
37. The method of claim 36 wherein the first flow rate is about 0.3
to 0.7 liters per minute.
38. The method of claim 20 wherein said detecting the selected
antigen bound to the antibody-bead conjugates by enzyme-linked
immunosorbent assay comprises: causing a medium comprising a
secondary antibody configured for binding the selected antigen to
flow through the module such that the secondary antibody binds to
the selected antigen bound to the antibody-bead conjugates; causing
a second wash medium to flow through the module such that secondary
antibody that did not bind to the selected antigen is washed out of
the module; causing a medium comprising a tertiary antibody-enzyme
conjugate configured for binding the secondary antibody to flow
through the module such that the tertiary antibody-enzyme conjugate
binds to the secondary antibody bound to the selected antigen;
causing a third wash medium to flow through the module such that
tertiary antibody-enzyme conjugate that did not bind to the
secondary antibody is washed out of the module; causing a medium
comprising an enzyme substrate to flow into the module, wherein the
enzyme substrate is selected for being a substrate for the tertiary
antibody-enzyme conjugate and being converted into a detectable
product, and incubating the enzyme substrate in the module such
that the detectable product is produced; and measuring the
detectable product.
39. The method of claim 38 wherein the detectable product is
luminescent.
40. The method of claim 38 wherein the detectable product is
fluorescent.
41. The method of claim 38 wherein the measuring the detectable
product is carried out with a photomultiplier tube.
42. An apparatus for use in capturing and detecting antigens
comprising: (a) a housing comprising a wall defining an interior
chamber and comprising an inlet opening for conducting a liquid
medium into the interior chamber and an outlet opening for
conducting the liquid medium out of the interior chamber, wherein
at least a portion of the wall is optically transparent; (b) at
least two antibody-bead conjugates disposed in the housing, each
comprising a bead, a polymeric spacer covalently coupled to the
bead, and an antibody coupled to the polymeric spacer; (c) a liquid
circulation circuit coupled to the housing for conducting the
liquid medium into the interior chamber through the inlet opening
and for conducting the liquid medium out of the interior chamber
through the outlet opening at a selected flow rate; and (d) a
photomultiplier tube mounted adjacent to the optically transparent
portion of the wall for measuring photons produced in the interior
chamber.
43. The apparatus of claim 42 wherein the bead is a glass bead.
44. The apparatus of claim 42 wherein the bead is a ceramic
bead.
45. The apparatus of claim 42 wherein the bead has a diameter of
about 1 to 7 millimeters.
46. The apparatus of claim 42 wherein the polymeric spacer
comprises dextran.
47. The apparatus of claim 42 wherein the polymeric spacer
comprises polyethylene glycol.
48. The apparatus of claim 42 wherein the polymeric spacer
comprises a polyamino acid.
49. The apparatus of claim 48 wherein the polyamino acid comprises
polythreonine.
50. The apparatus of claim 48 wherein the polyamino acid comprises
polyserine.
51. The apparatus of claim 42 wherein the antibody comprises a
monoclonal antibody.
52. The apparatus of claim 42 wherein the antibody comprises a
polyclonal antibody.
53. The apparatus of claim 42 wherein the antibody comprises an
antibody fragment.
54. The apparatus of claim 42 wherein the liquid circulation
circuit comprises a pump.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/163,253, filed Jun. 4, 2002, abandoned, which is a
continuation-in-part of U.S. Ser. No. 09/292,172, filed Apr. 15,
1999, now U.S. Pat. No. 6,399,317, which claims the benefit of U.S.
Provisional Application No. 60/081,889, filed Apr. 15, 1998, all of
which are hereby incorporated by reference in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] This invention relates to detection of antigens. More
particularly, the invention relates to compositions and methods for
detection of selected antigens in real time. In an illustrative
embodiment, the invention relates to compositions and processes for
sensitive detection of microbes and contaminants in complex
samples, such as food samples, environmental samples, and the like,
within about 30 minutes.
[0004] Bacterial spores are the most heat-stable form of
microorganisms, are ubiquitous in the environment, and are
therefore of great concern in food products, such as milk, that
receive extensive heat treatments to prolong shelf life. Spore
counts in milk from around the world vary from zero to greater than
22,000 cfu/ml depending on the climate of the region. S. A. Chen et
al., A Rapid, Sensitive and Automated Method for Detection of
Salmonella Species in Foods using AG-9600 AmpliSensor Analyzer, 83
J. Appl. Microbiol. 314-321 (1997). Bacillus stearothermophilus
spores are one of the most heat-resistant bacterial spores and are
found in high numbers in soil and water. Contaminating B.
stearothermophilus spores survive extreme heat to germinate and
grow at elevated product storage temperatures, which occur in foods
transported in equatorial regions of the world.
[0005] While B. stearothermophilus is not commonly a problem, other
bacilli often lead to food-borne illness or spoilage in a variety
of foods. Bacillus cereus, Bacillus licheniformis, Bacillus
subtilis, and Bacillus pumilus have all been implicated in
outbreaks of food-borne illness and are commonly isolated from raw
and heat treated milk. M. W. Griffiths, Foodborne Illness Caused by
Bacillus spp. other than B. cereus and Their Importance to the
Dairy Industry, 302 Int. Dairy Fed. Bulletin 3-6 (1995). B. cereus
is also responsible for a sweet curdling defect in milk as well as
being pathogenic. W. W. Overcast &K. Atmaram, The Role of
Bacillus cereus in Sweet Curdling of Fluid Milk, 37 J. Milk Food
Technol. 233-236 (1973). A mesophilic heat resistant bacillus
similar to Bacillus badius, has been isolated from extreme
temperature processed milk (D.sub.147=5 sec; P. Hammer et al.,
Pathogenicity Testing of Unknown Mesophilic Heat Resistant Bacilli
from UHT-milk, 302 Int. Dairy Fed. Bulletin 56-57 (1995)). B.
badius is a mesophilic organism and grows readily at room
temperature, making it a likely candidate for spoiling
temperature-processed foods. There have been 52 confirmed cases of
B. badius in UHT milk in Europe and two cases outside of Europe. P.
Hammer et al., supra. Lack of a rapid spore assay that can be used
in milk contributes to the difficulty of prediction of post
processing spoilage, thereby limiting the shelf life and product
safety. H. Hofstra et al., Microbes in Food-processing Technology,
15 FEMS Microbiol. Rev. 175-183 (1994). Such an assay could be used
in a hazard analysis critical control point (HACCP) plan allowing
raw materials with high spore loads to be diverted to products that
do not pose a food safety risk to consumers.
[0006] The standard method for quantifying spores in milk involves
heat-shocking and an overnight plate count. G. H. Richardson,
Standard Methods for the Examination of Dairy Products (1985). This
is time-consuming and yields historical information. The food
industry needs microbiological assays to yield predictive
information for maximum benefit in HACCP analysis and risk
assessment. An enzyme-linked immunosorbent assay (ELISA) capable of
detecting greater than 10.sup.6 cfu/ml of B. cereus spores in foods
has been reported, but was unacceptable due to antibody
cross-reactivity. Y. H. Chang & P. M. Foegeding, Biotin-avidin
Enzyme-linked Immunosorbent Assay for Bacillus Spores in Buffer and
Food, 2 J. Rapid Methods and Autom. Microbiol. 219-227 (1993).
[0007] Techniques to increase sensitivity of immunosorbent assays
have focused on more efficient reporter labels, such as faster
catalyzing reporter-enzymes; signal amplification, such as avidin-
or streptavidin-biotin enzyme complexes; and better detectors, such
as luminescence and fluorescence. L. J. Kricka, Selected Strategies
for Improving Sensitivity and Reliability of Immunoassays, 40 Clin.
Chem. 347-357 (1994); P. Patel, Rapid Analysis Techniques in Food
Microbiology (1994). Immunomagnetic antigen capture is used
extensively to separate and identify Escherichia coli and
Salmonella from foods. C. Blackburn et al., Separation and
Detection of Salmonellae Using Immunomagnetic Particles, 5
Biofouling 143-156 (1991); P. M. Fratamico et al., Rapid Isolation
of Escherichia coli O157:H7 from Enrichment Cultures of Foods Using
an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113
(1992); L. Krusell & N. Skovgaard, Evaluation of a New
Semi-automated Screening Method for the Detection of Salmonella in
Foods within 24 h, 20 Inter. J. Food Microbiol. 124-130 (1993); A.
Lund et al., Rapid Isolation of K88.sup.+ Escherichia coli by Using
Immunomagnetic Particles, 26 J. Clin. Microbiol. 2572-2575 (1988);
L. P. Mansfeild & S. J. Forsythe, Immunomagnetic Separation as
an Alternative to Enrichment Broths for Salmonella Detection, 16
Letters Appl. Microbiol. 122-125 (1993); A. J. G. Okrend et al.,
Isolation of Escherichia coli O157:H7 Using O157 Specific Antibody
Coated Magnetic Beads, 55 J. Food Prot. 214-217 (1992); E. Skjerve
& Olsvic, Immunomagnetic Separation of Salmonella from Foods,
14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright et al.,
Immunomagnetic Separation as a Sensitive Method for Isolating
Escherichia coli O157 from Food Samples, 113 Epidemiol. Infect.
31-39 (1994). However, these methods involve either a preincubation
or a subsequent incubation step (usually 18 to 24 hours) to
increase the cell numbers for detection. Immunomagnetic capture
greatly shortens E. coli and Salmonella testing, but long
incubation times limit this method for predictive information.
Immunocapture has also been used to quantitate Bacillus anthracis
spores in soil samples using luminescent detection, J. G. Bruno
& H. Yu, Immunomagnetic-electrochemiluminescent Detection of
Bacillus anthracis Spores in Soil Matrices, 62 App. Environ.
Microbiol. 3474-3476 (1996), but these efforts have led to tests
that have a detection limit of 10.sup.3 cfu/ml.
[0008] Considerable progress in the development of biosensors for
microbial detection has been achieved in the last decade. These
biosensors can be applied to medical, process control, and
environmental fields. They must possess ideal features such as high
specificity, simplicity, sensitivity, reliability, reproducibility,
and speed. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit.
Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as
the recognition element for specific capture, numerous applications
have been developed for detection of pathogenic bacteria. M. R.
Blake & B. C. Weimer, Immunomagnetic Detection of Bacillus
stearothermophilus Spores in Food and Environmental Samples, 63 J.
Appl. Environ. Microbiol. 1643-1646 (1997); A. Burkowski, Rapid
Detection of Bacterial Surface Proteins Using an Enzyme-linked
Immunosorbent Assay System, 34 J. Biochem. Biophys. Methods 69-71
(1997); S. A. Chen et al., A Rapid, Sensitive and Automated Method
for Detection of Salmonella Species in Foods Using AG-9600
AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L. A.
Metherell et al., Rapid, Sensitive, Microbial Detection by Gene
Amplification using Restriction Endonuclease Target Sequence, 11
Mol. Cell Probes 297-308 (1997); F. Roth et al., A New Multiantigen
Immunoassay for the Quantification of IgG Antibodies to Capsular
Polysaccharides of Streptococcus pneumoniae, 176 J. Inf. Dis.
526-529 (1997).
[0009] Methods for continuous flow immunoassay for rapid and
sensitive detection of small molecules have been developed. For
example, A. W. Kusterbeck et al., 135 J. Immunol. Methods 191-197
(1990), describes such a method in which detection of the antigen
occurred within a matter of minutes. The assay is based on the
binding of labeled antigen to an immobilized antibody, with
subsequent displacement of the labeled antigen when antigen is
present in the sample flow. Signal detection occurs downstream of
the antigen recognition event.
[0010] In standard displacement flow immunoassays, the analyte of
up to 1000 molecular weight in the sample creates an active
dissociation of labeled antigens from an antigen binding site of an
immobilized antibody, after which the labeled substance is measured
downstream. W. A. Kaptein et al., On-line Flow Displacement
Immunoassay for Fatty Acid-binding Protein, 217 J. Immunol. Methods
103-111 (1998), describes displacement in a flow system for
detection of a small protein, cytoplasmic heart-type fatty
acid-binding protein (15,000 molecular weight), a plasma marker for
myocardial injury. This displacement system uses an inverse set-up:
enzyme-labeled monoclonal antibodies are associated to immobilized
antigen and are displaced by analyte in the sample.
[0011] F. Vianello et al., Continuous Flow Immunosensor for
Atrazine Detection, 13 Biosens. Bioelectron. 45-53 (1998),
describes detection of the hapten, atrazine, under continuous flow
conditions using a micro-column containing immobilized monoclonal
antibodies against atrazine and atrazine labeled with alkaline
phosphatase. The equilibrium of the antibody-hapten system was
achieved by continuous flow of the tracer (alkaline
phosphatase-labeled atrazine) through the micro-column containing
the immobilized antibodies. The activity of the tracer was
monitored continuously downstream of the micro-column with an
amperometric detector using p-hydroquinone phosphate as substrate.
When pulses of unlabeled atrazine were added to the tracer flowing
continuously through the micro-column, tracer bound to the antibody
was displaced, with a consequent change in the detector signal.
[0012] C. H. Pollema & J. Ruzicka, Flow Injection Renewable
Surface Immunoassay: A New Approach to Immunoanalysis with
Fluorescence Detection, 66 Anal. Chem. 1825-1831 (1994), describes
automatic heterogeneous immunoassays using a flow injection
technique on a renewable surface. This assay relies on a minute
amount of beads to form a reactive surface, which is interrogated
by fluorescence spectrometry. Following the assay, the spent
reactive surface is fluidically removed and replaced with a new
layer of beads.
[0013] B. Mattiasson & M. P. Nandakumar, Binding Assays in
Heterogeneous Media Using a Flow Injection System with an Expanded
Micro-bed Adsorption Column, 8 Bioseparation 237-245 (1999),
describes a competitive binding assay in a flow injection system
wherein the adsorption step was carried out in an expanded bed
column to increase the versatility of the assay an enable it to
deal with samples containing particulate matter.
[0014] In view of the foregoing, it will be appreciated that
compositions and methods for real time detection of selected
antigens, such as contaminants in food and the environment, would
be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
[0015] The present invention comprises compositions and methods for
capture and detection of antigens from complex liquid samples
within a matter of minutes and without the need for culturing of
organisms.
[0016] An illustrative method according to the present invention
for capturing and concentrating a selected antigen from an aqueous
medium containing a mixture of antigens comprises:
[0017] (a) causing a first volume of the aqueous medium containing
the mixture of antigens to flow through a module containing at
least two antibody-bead conjugates, wherein each of the
antibody-bead conjugates comprises a bead, a polymeric spacer
covalently coupled to the bead, and an antibody covalently coupled
to the polymeric spacer, wherein the antibody is configured for
binding the selected antigen, at a first flow rate such that the
antibody-bead conjugates form a fluidized bed and the selected
antigen binds to the antibody-bead conjugates;
[0018] (b) washing the antibody-bead conjugates having the selected
antigen bound thereto by causing a wash medium to flow through the
module at a second flow rate such that the antibody-bead conjugates
having the selected antigen bound thereto form a fluidized bed;
and
[0019] (c) holding the washed antibody-bead conjugates having the
selected antigen bound thereto in a second volume of a second wash
medium, wherein the second volume is smaller than the first
volume.
[0020] Another illustrative method according to the present
invention for detecting a selected antigen in aqueous medium
containing a mixture of antigens comprises:
[0021] (a) causing the aqueous medium containing the mixture of
antigens to flow through a module containing at least two
antibody-bead conjugates, wherein each of the antibody-bead
conjugates comprises a bead, a polymeric spacer covalently coupled
to the bead, and an antibody covalently coupled to the polymeric
spacer, wherein the antibody is configured for binding the selected
antigen, at a first flow rate such that the antibody-bead
conjugates form a fluidized bed and the selected antigen binds to
the antibody-bead conjugates;
[0022] (b) washing the antibody-bead conjugates having the selected
antigen bound thereto by causing a first wash medium to flow
through the module at a second flow rate such that the
antibody-bead conjugates having the selected antigen bound thereto
form a fluidized bed; and
[0023] (c) detecting the selected antigen bound to the
antibody-bead conjugates by enzyme-linked immunosorbent assay.
[0024] An illustrative apparatus according to the present invention
for use in capturing and detecting antigens comprises:
[0025] (a) a housing comprising a wall defining an interior chamber
and comprising an inlet opening for conducting a liquid medium into
the interior chamber and an outlet opening for conducting the
liquid medium out of the interior chamber, wherein at least a
portion of the wall is optically transparent;
[0026] (b) at least two antibody-bead conjugates disposed in the
housing, each comprising a bead, a polymeric spacer covalently
coupled to the bead, and an antibody coupled to the polymeric
spacer;
[0027] (c) a liquid circulation circuit coupled to the housing for
conducting the liquid medium into the interior chamber through the
inlet opening and for conducting the liquid medium out of the
interior chamber through the outlet opening at a selected flow
rate; and
[0028] (d) a photomultiplier tube mounted adjacent to the optically
transparent portion of the wall for measuring photons produced in
the interior chamber.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0029] FIG. 1A illustrates specific capture of B.
stearothermophilus spores from a mixed population containing equal
amounts of B. stearothermophilus and B. subtilis spores, where B.
subtilis spore counts in PBST are represented by the dotted bar, B.
stearothermophilus spore counts in PBST are represented by the open
bar, B. subtilis spore counts in milk are represented by the
hatched bar, and B. stearothermophilus spore counts in milk are
represented by the solid bar. FIG. 1B shows the concentration of
spores in the wash: (.quadrature.) B. subtilis in PBST, (.diamond.)
B. stearothermophilus in PBST, (.smallcircle.) B. subtilis in milk,
and (.DELTA.) B. stearothermophilus in milk.
[0030] FIG. 2 shows fluorescence detection of captured B.
stearothermophilus spores in skim milk by a biotin-avidin amplified
sandwich ELISA using 3.times.10.sup.6 (.quadrature.) and
1.4.times.10.sup.7 (.smallcircle.) immunomagnetic beads (IMBs);
data points represent the mean of two replications, and error bars
represent standard error of the means.
[0031] FIG. 3 shows fluorescence detection of captured B.
stearothermophilus spores from various food and environmental
samples using 3.times.10.sup.6 IMBs: (.diamond.) muck clay,
R.sup.2=0.82; (.smallcircle.) pepper, R.sup.2=0.96; (.quadrature.)
skim milk, R.sup.2=0.99; (.DELTA.) whole milk; (.box-solid.) acidic
sandy soil (pH 3.7); data points represent the mean of two
replications, and error bars represent standard error of the
means.
[0032] FIG. 4 shows a schematic representation of an illustrative
module for use in detecting antigens according to the present
invention.
[0033] FIG. 5 shows immunoflow (2 L/min) detection of B. globigii
spores in 0.1 M phosphate buffer (pH 7.2, R.sup.2=0.9;
slope=0.03).
[0034] FIG. 6 shows a bovine serum albumin (BSA) standard curve at
the range of 1.5-100 ng/.mu.l; each point is the average absorbance
(450 nm) in triplicate, and PBST (0.2% Tween 20) was the blocking
reagent to block the nonspecific binding sites.
[0035] FIG. 7 shows a BSA standard curve at the range of 0.075-2.5
ng/.mu.l; each point is the average absorbance (450 nm) in
triplicate; PBST (0.2% Tween 20) was the blocking reagent.
[0036] FIG. 8 shows a schematic diagram of an illustrative
composition for use in binding antigens to a solid support
according to the present invention.
[0037] FIG. 9 shows a schematic diagram of detection of antigens
captured with the composition of FIG. 8.
[0038] FIGS. 10 and 11 show a module or bead container for holding
the bead-bound antibodies for capturing and detecting antigens
according to the present invention. FIG. 10 shows the beads settled
by gravity at the bottom of the module, whereas FIG. 11 shows the
positions of beads in a fluidized condition when liquid is passing
through the module.
[0039] FIG. 12 shows a schematic diagram of an illustrative
apparatus for use in detecting antigens according to the present
invention.
[0040] FIG. 13. shows capture of E. coli O157:H7 from buffer on the
surface of bead-bound antibodies.
[0041] FIG. 14 shows the average capture efficiency E. coli O157:H7
from buffer, beer, and apple juice using bead-bound antibodies.
[0042] FIG. 15 shows signal detection from the surface of
beads.
[0043] FIG. 16 shows determination of cell density by the method of
the present invention.
[0044] FIGS. 17A-C show calibration plots of the relative capture
activity versus concentration of antigen. Two types of glass beads,
succinylated (.circle-solid.) and PEG-coupled (.box-solid.), were
used. FIG. 17A shows results for ovalbumin (OVA). FIG. 17B shows
results for B. globigii spores. FIG. 17C shows results for E. coli
O157:H7.
[0045] FIG. 18 shows a standard curve for BSA capture with
tosyl-activated, polythreonine-modified immunomagnetic beads.
Standard error of the mean at each data point is masked by the
symbol.
[0046] FIG. 19 shows a standard curve of OVA using 3 mm
PEG-modified glass beads. Error bars represent standard error of
the mean.
[0047] FIGS. 20A&B show standard curves of B. globigii spores
in PBST (FIG. 20A) and E. coli O157:H7 in meat extract
(.quadrature.) and PBST (.box-solid.) (FIG. 20B) using 3 mm
PEG-modified glass beads. Standard error of the mean for each data
point is masked by the symbol.
[0048] FIG. 21 shows standard curves of B. globigii spores in
environmental and industrial water samples using PEG-modified glass
beads: PBST (.box-solid.), river water (.diamond-solid.), tank
water (.smallcircle.), slush tank (.DELTA.). Error bars represent
standard error of the mean.
DETAILED DESCRIPTION
[0049] Before the present compositions and methods for real time
detection of antigens are disclosed and described, it is to be
understood that this invention is not limited to the particular
configurations, process steps, and materials disclosed herein as
such configurations, process steps, and materials may vary
somewhat. It is also to be understood that the terminology employed
herein is used for the purpose of describing particular embodiments
only and is not intended to be limiting since the scope of the
present invention will be limited only by the appended claims and
equivalents thereof.
[0050] The publications and other reference materials referred to
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0051] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise. For
example, reference to an apparatus containing "a bead" includes
reference to two or more of such beads, reference to "a spacer"
includes reference to one or more of such spacers, and reference to
"an antibody" includes reference to two or more of such
antibodies.
[0052] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out herein.
[0053] As used herein, "antibody" means an immunoglobulin molecule
that interacts and binds only with the antigen that induced its
synthesis in lymphoid tissue and/or with antigens closely related
to it. Included within this definition of antibody all antibody
types, e.g., IgG, IgA, IgM, etc.; IgG subclasses, e.g., IgG1, IgG2,
etc.; F(ab) fragments; F(ab').sub.2 fragments; F(ab') fragments;
light chain dimers; single chain antibodies, and the like. This
definition also includes antibodies that react with low and high
affinity with an antigen. Further, such antibodies can be
polyclonal or monoclonal.
[0054] As used herein, "cfu" means colony forming units.
[0055] As used herein, "PBS" means phosphate buffered saline: 0.01
M Na.sub.2HPO.sub.4, 0.15 M NaCl, pH 7.2.
[0056] As used herein, "PBST" means phosphate buffered saline
containing 0.1% Tween 20.
[0057] As used herein, "BSA" means bovine serum albumin.
[0058] As used herein, "ELISA" means enzyme-linked immunosorbent
assay.
[0059] As used herein, "comprising," "including," "containing,"
"characterized by," and grammatical equivalents thereof are
inclusive or open-ended terms that do not exclude additional,
unrecited elements or method steps. "Comprising" is to be
interpreted as including the more restrictive terms "consisting of"
and "consisting essentially of."
[0060] As used herein, "consisting of" and grammatical equivalents
thereof exclude any element, step, or ingredient not specified in
the claim.
[0061] As used herein, "consisting essentially of" and grammatical
equivalents thereof limit the scope of a claim to the specified
materials or steps and those that do not materially affect the
basic and novel characteristic or characteristics of the claimed
invention.
[0062] A fluidized or extended volume reactor filled with beads
that have been modified with antibodies was developed for capturing
antigens, such as specific microorganisms and biological molecules.
The beads can be glass, ceramic, and the like, and are relatively
large compared to those used in many antibody capture processes,
typically in the range of about 1-7 millimeters (mm) in diameter.
The antibodies are attached to the beads through a spacer. Typical
spacers include polymers such as dextran, polyethylene glycol
(PEG), and polyamino acids, such as polyserine and
polythreonine.
[0063] Flow speed is typically about 0.2 to about 1.2 liters per
minute. More typically, the flow speed is about 0.3 to about 0.7
liters per minute. The flow of the sample and wash solutions
through the bead-containing module can be generated by use of
vacuum pump, peristaltic pump, and other similar methods known in
the art.
[0064] Detection of captured antigens can be by methods well known
in the art, such as by surface ELISA using chemiluminescence
(photometric), fluorescence, and spectrophotometric detection.
[0065] Luminescence is related to fluorescence in that both produce
photons or light. In the case of fluorescence, however, energy must
also be applied to excite the photons to escape the molecular
structure. This is most often done with a laser and specific
wavelengths of light. Luminescence does not require light input
since chemicals or biological molecules provide the energy to
excite the molecule. Unlike detection systems based on
fluorescence, chemiluminescence methods do not require external
light sources for excitation energy. The signals are generated
internally as light-producing chemical reactions occur.
[0066] Detection of antigens according to the present invention
typically involves a luminescence reaction, although fluorescence
or colorimetric detection can also be used. The reporter enzyme
used in the reaction determines which chemiluminescent substrate is
employed. Horseradish peroxidase (HRP) and alkaline phosphatase
(AP) are the two most common reporter enzymes. A common substrate
is luminol (cyclic diacyl hydrazide), which is oxidized during the
enzyme reaction. This oxidation converts the luminol substrate into
an excited intermediate dianion. As the intermediate returns to its
ground state, it emits light at a maximum of 425 nm. Another
substrate is Lumigen APS-5 (Lumigen, Inc., Southfield, Mich.),
which emits light at a maximum of 430 nm. Chemiluminescence is
typically about 2 orders of magnitude more sensitive than
fluorescence and more than 4 orders of magnitude more sensitive
than chromogenic reactions. This sensitivity allows for lower
detection limits in standard assays, such as ELISA.
EXAMPLE 1
[0067] Bacteria.
[0068] The bacteria used in the experiments described herein are
described in Table 1. Commercial preparations of spores of B.
stearothermophilus ATCC 10149, B. cereus ATCC 11778, and B.
subtilis 6633 were purchased from Fisher Scientific, Pittsburgh,
Pa. Viable spore numbers and germination estimates were obtained by
plating on plate count agar (PCA) overnight at 65.degree. C. and
30.degree. C., respectively. All other spores except for B.
globigii spores (Table 1) were prepared by spread-plating a single
colony isolate on PCA and incubating the covered plate at
30.degree. C. for approximately 2 weeks. Spores were swabbed from
the surface of the agar and washed repeatedly in distilled water to
remove water-soluble components. Spores were pelleted and separated
from cell debris by centrifugation (1,500.times.g for 20 min at
4.degree. C.; D. E. Gombas & R. F. Gomez, Sensitization of
Clostridium perfringens Spores to Heat by Gamma Radiation, 36 Appl.
Environ. Microbiol. 403-407 (1987)). Presence of spores was
confirmed by heating to 80.degree. C. for 15 min and then plating
on PCA (G. H. Richardson, supra). Presence of an exosporium on the
spore was tested by phase contrast microscopy with crystal violet
staining (C. Du & K. Nickerson, Bacillus thuringiensis HD-73
Spores Have Surface-localized Cry Ac Toxin: Physiological and
Pathogenic Consequences, 62 Appl. Environ. Microbiol. 3722-3726
(1996)). Spore titers were estimated by plating spores on plate
count agar (PCA) and incubating overnight at 37.degree. C. Based on
these experiments, it was estimated that 10.sup.11 spores have a
mass of 1 g.
1TABLE 1 Inc. temp. Species (.degree. C.) Source Exosporium B.
stearothermophilus 65 ATCC 10149.sup.a - B. cereus 30 ATCC
11778.sup.a + B. subtilis 30 ATCC 6633.sup.b + B. circulans 30 ATCC
4513.sup.b - B. coagulans 30 ATCC 7050.sup.b - B. globigii 30
Dugway.sup.c B. licheniformis 30 OSU.sup.d - B. mascerans 30
OSU.sup.d + B. polymyxa 30 ATCC 842.sup.b + B. pumilus 30 OSU.sup.d
- .sup.aPurchased from Fisher Scientific, Pittsburgh, Pennsylvania.
.sup.bPurchased from American Type Culture Collection.
.sup.cObtained from Dugway Proving Grounds (Tooele, Utah).
.sup.dDonated by Floyd Bodyfelt, Oregon State University.
EXAMPLE 2
[0069] Polyclonal Antibodies Production.
[0070] Polyclonal antibodies against B. cereus spores, B. subtilis
spores, and B. stearothermophilus spores were made at the Utah
State Biotechnology Center (Logan, Utah). BALB/c mice were injected
in the intraperitoneal cavity with 1.times.10.sup.7 cfu/ml cells or
spores in sterile saline (0.5 ml) three times at 3-week intervals.
E. Harlow & D. Lane, Antibodies, A Laboratory Manual (1988).
Total ascites IgG was purified using a protein A/G column (Pierce
Chemical, Rockford, Ill.). Antibodies were desalted and
concentrated to 1 mg/ml in 0.1 M NaPO.sub.4, pH 7.0 in a 30 kD
Centricon filter (Amicon, Beverly, Mass.) at 4,500.times.g at
4.degree. C.
[0071] Goat antibodies to Bacillus globigii spores were obtained
from Dugway Proving Grounds (Tooele, Utah).
EXAMPLE 3
[0072] Monoclonal Antibody Production.
[0073] Monoclonal antibodies were produced against B.
stearothermophilus by suspending the cells or spores in PBS to an
optical density of 0.93 at 550 nm before intraperitoneally
injecting female BALB/c mice with 0.250 mg (whole cell wet weight)
without adjuvant. The mice were immunized 3 times at 3-week
intervals. Seven days after the last immunization they were test
bled, and the serum was titered by ELISA 3 days before fusion.
Booster injections were administered by intraperitoneal injection
with 0.1 mg cells in PBS. Fusion with a compatible murine myeloma
cell line (P3X63-Ag8.653) was done in the presence of polyethylene
glycol. Selection for hybrid cells was done in HAT medium. G.
Kohler & C. Milstein, Continuous Cultures of Fused Cells
Secreting Antibody of Pre-defined Specificity, 256 Nature 495-97
(1975) (hereby incorporated by reference). Positive colonies were
determined by ELISA and were subcloned twice before freezing in
liquid nitrogen.
EXAMPLE 4
[0074] Antibody Specificity.
[0075] Antibody specificity was tested by measuring the cross
reactivity against Bacillus spores listed in Table 1 using a
standard ELISA. A suspension of each spore type (10.sup.6 cfu/ml),
suspended in 50 mM NaCO.sub.3 (pH 9.5), was nonspecifically bound
to wells of a microtiter plate for 12 h at 4.degree. C. Wells
containing spores were blocked with 2% bovine serum albumin (BSA)
in PBS for 4 h at 25.degree. C., and washed four times with PBS
containing 0.1% Tween 20 (PBST). Anti-B. stearothermophilus
antibodies (1:10,000 serum dilution in PBS) were added to wells,
slowly agitated for 2 h at 25.degree. C., and washed four times
with PBST. Horseradish-peroxidase-labeled (HRP) anti-whole mouse
IgG (Sigma Chemical Co., St. Louis, Mo.) was added to label anti-B.
stearothermophilus antibodies for 2 h, then the wells were washed
four times with PBST. O-Phenylenediamine dihydrochloride (Sigma)
color development was measured using a b* color scale (blue to
yellow) at 37.degree. C. for 1 h in an automated reflectance
calorimeter (Omnispec 4000 Bioactivity monitor; Wescor, Inc.,
Logan, Utah).
[0076] The anti-B. stearothermophilus antibodies did not cross
react with any of the spore types tested (Table 1) including common
aerobic spores found in raw foods. Lack of cross reactivity may be
partly due to the absence of an exosporium on the B.
stearothermophilus spores (Table 1). However, antibodies raised
against B. subtilis and B. cereus, which have exosporia, were also
specific for the injected spore types, suggesting that the surface
antigens of the exosporia are sufficiently different as to not
crossreact.
EXAMPLE 5
[0077] Antibody Attachment to Beads Via Biotin-Streptavidin.
[0078] Anti-B. stearothermophilus antibodies purified from total
serum were biotinylated with NHS-LC-Biotin (Pierce Chemical,
Rockford, Ill.). Efficiency of surface biotinylation was determined
using the HABA assay (Pierce), except that the 2-mercaptoethanol
step was omitted to avoid denaturing antibodies. This modified
procedure gave the number of surface biotin moieties per antibody
(Sigma Technical Support).
[0079] Biotinylated antibodies were coupled to streptavidin-bound
magnetic beads (Dynabeads Streptavidin.TM., Lake Success, N.Y.)
according to the supplier's directions.
EXAMPLE 6
[0080] Antibody Attachment to Bead Via Poly(threonine).
[0081] Sodium meta-periodate (5 mg) was used to oxidize
carbohydrate moieties on the anti-B. stearothermophilus antibodies.
G. T. Hermanson et al., Immobilized Affinity Ligand Techniques
(1992) (hereby incorporated by reference). Sodium meta-periodate
was removed after oxidation by washing five times with 0.1M
NaPO.sub.4, pH 7.0, in a 30 kD Centricon filter (4,500.times.g,
4.degree. C.), and the oxidized antibodies were then immediately
crosslinked to beads magnetic beads.
[0082] PolyThr (MW(vis) 12,100;Sigma Chemical, St. Louis, Mo.) was
covalently coupled to 2.8-.mu.m, tosyl-activated polystyrene
Dynabeads (Dynal, Lake Success, N.Y.) in 50 mM borate buffer (pH
9.5) via the terminal amine as described by the product
instructions. Four washes (three times for 10 min, and once for 30
min) with TBS buffer (pH 7.5) were used to block remaining
tosyl-active sites. Adenine dihydrazine (ADH; 0.5 M in 0.1 M MES,
pH 4.75; Sigma) was linked to the carboxy terminus of the bound
PolyThr using an ethylene diamine carbodiimide mediated reaction
(G. T. Hermanson et al., supra). Oxidized antibodies were mixed
with the ADH-activated beads at room temperature for 12 h to allow
crosslinking between the oxidized carbohydrate moiety of the IgG
and the ADH terminus of the PolyThr spacer (G. T. Hermanson et al.,
supra). After crosslinking, the modified immunomagnetic beads
(IMBs) were stored rotating (50 rpm) in PBST with 0.02% sodium
azide at 40.degree. C. until use.
EXAMPLE 7
[0083] Antibody Attachment to Bead Via Poly(serine).
[0084] In this example, the procedure of Example 6 was followed
except that poly(serine) was substituted for poly(threonine).
EXAMPLE 8
[0085] Antibody Attachment to Bead Via Dextran.
[0086] Ceramic beads, 7 mm in diameter (Coors Ceramics Corp.,
Golden, Colo.), were washed in acidic methanol (HCl:methanol, 1:1)
for 30 min at room temperature (RT) to strip the bead surface. The
acidic methanol was poured off and the beads were rinsed several
times with filtered water (dH.sub.2O). The beads were further
washed with concentrated sulfuric acid three times for 30 min,
rinsed several times with dH.sub.2O, and finally boiled in
dH.sub.2O for 30 min to introduce hydroxyl groups onto the
surface.
[0087] For silanization and crosslinking, beads were air dried,
washed once in toluene and incubated in 3% 3-mercapto propyl
trimethoxysilane (3% MTS in toluene) for 2 h at RT. Subsequently
the beads were prepared for the addition of the crosslinker
.gamma.-maleimidobutyric acid N-hydroxy succinimide ester (GMBS;
Sigma Chemicals, St. Louis, Mo.). Beads were washed twice in
toluene to remove unbound MTS, air dried, and then incubated for 1
h at RT in 2 mM GMBS (in 100% ethanol). Finally, the beads were
finally washed in 100% ethanol and then in PBS.
[0088] Dextran was used as a spacer between the crosslinker and the
antibody. Sodium-m-periodate (Sigma Chemicals, St. Louis, Mo.) was
used to oxidize the carbohydrate moieties on dextran (37.5 kDa,
Sigma Chemicals, St. Louis, Mo.) for 3 h at RT while shaking. The
salt was removed by washing four times with dH.sub.2O in 30 kDa
Centricon filters (Amicon Inc., Beverly, Mass.) and immediately
bound to the crosslinked beads. Adipic acid dihydride (ADH, 0.5 M
in sodium phosphate, pH 7.2;Sigma Chemicals, St. Louis, Mo.) was
then added to introduce an amine group to the bead surface, which
could then react with the oxidized antibody. All unreacted sites
were blocked with 1% Tris/BSA, pH 8.5.
EXAMPLE 9
[0089] In this example, anti-B. stearothermophilus antibodies were
mixed with tosyl-activated magnetic beads (Dynal) according to the
directions supplied with the beads such that the amine groups on
the antibodies reacted with tosyl groups on the surface of the
beads. The resulting modified beads contained the antibodies
covalently bonded to the surface of the beads.
EXAMPLE 10
[0090] In this example, anti-Fc IgG was bound to magnetic beads
according to the procedure of Example 9. After unbound IgG was
washed off, the beads were reacted with anti-B. stearothermophilus
antibodies such that the anti-Fc IgG bound the anti-B.
stearothermophilus antibodies. The anti-Fc IgG thus formed a spacer
between the magnetic beads and the anti-B. stearothermophilus
antibodies.
EXAMPLE 11
[0091] In this example, anti-B. stearothermophilus antibodies
attached to magnetic beads were tested for their ability to capture
B. stearothermophilus spores. The antibody/bead conjugates (i.e.,
immunomagnetic beads or IMBs) were prepared according to the
procedure of Examples 5, 6, 9, and 10. ELISA using HRP-labeled
anti-IgG confirmed the presence of bound antibodies on the surfaces
of the beads.
[0092] IMBs (3.times.10.sup.6 beads) were added to 1 ml of sample
comprising 10.sup.4 or 10.sup.6 B. stearothermophilus spores in
PBST. These mixtures were incubated for 30 min at 25.degree. C.
with rotation at 50 rpm. The IMBs were removed from the sample for
2 min with a magnetic particle concentrator (Dynal MPC-E-1) and
washed four times with PBST to reduce IMB clumping and block spore
adhesion to tube walls (E. Skjerve et al., Detection of Listeria
monocytogenes in Foods by Immunomagnetic Separation, 56 Appl.
Environ. Microbiol. 3478-3481 (1990)). After each wash, IMBs were
transferred to a new microfuge tube. The presence of bound spores
on IMBs was confirmed in duplicate by plate counts and phase
contrast microscopy.
2TABLE 2 Table 2 shows the results of these experiments. Ab No.
spores Attachment Modification Ab Orientation bound Biotin- NHS-LC-
Non- 0.sup.a Streptavidin Biotinylation directional 0.sup.b
Ab-NH.sub.2 to None Non- 0.sup.a Tosyl groups directional 0.sup.b
on beads Anti-Fc IgG None Directional 0.sup.a spacer 0.sup.b
PolyThr-ADH Carbohydrate Directional 160.sup.a crosslinker
oxidation 3600.sup.b .sup.aCaptured from 10.sup.4 spores/ml.
.sup.bCaptured from 10.sup.6 spores/ml.
[0093] These results show that of the conjugates tested only
antibodies bound to beads through a poly(threonine) spacer were
able to capture spores. These data suggest that spacer length and
flexibility may play a role in the antibody-antigen
interaction.
EXAMPLE 12
[0094] In this example, the procedure of Example 11 was followed
except that conjugates containing poly(serine) (Example 7) and
dextran (Example 8) spacers were substituted for the conjugate
containing the poly(threonine) spacer. The results obtained with
the poly(serine)--and dextran-containing conjugates were
substantially similar to those obtained with the
poly(threonine)--containing conjugate.
EXAMPLE 13
[0095] In this example, equal numbers of B. subtilis and B.
stearothermophilus spores were mixed in PBST and in milk.
Immunocapture using anti-B. stearothermophilus antibodies
conjugated to magnetic beads was carried out according to the
procedure of Example 11 except that samples containing milk were
given 5 minutes to separate the beads from the medium using the
magnetic particle concentrator due to the slower bead recovery.
After capture of spores using the immunomagnetic bead conjugate,
the conjugates were washed with PBST and the wash supernates were
plated on PCA. This washing procedure was repeated three times such
that a total of four wash supernates were assayed.
[0096] FIG. 1A shows the conjugate specifically captured B.
stearothermophilus spores from PBST and milk containing equal
numbers of B. stearothermophilus and B. subtilis spores. FIG. 1B
shows that about 99% of non-specifically bound spores were removed
from the conjugate with each wash, leaving B. stearothermophilus
spores captured by the conjugate after four washes.
[0097] The anti-B. stearothermophilus antibodies did not cross
react with any of the spore types tested (Table 1) including common
aerobic spores found in raw foods.
EXAMPLE 14
[0098] Product Testing.
[0099] In this example, muck clay, ground pepper, skim milk, whole
milk, and acidic sandy soil were tested for detection of bacterial
spores. Fluid products were tested with no modification. Powdered
products were suspended at 1 g/ml. Anti-B. stearothermophilus
antibody conjugated beads prepared according to the procedure of
Example 6 were added to 1 ml of each product and mixed gently at
25.degree. C. Bound spores were quantitated using calorimetric (as
described above) or fluorescence detection. For fluorescence
detection, spores bound to IMBs were labeled with secondary
biotinylated anti-B. stearothermophilus antibodies. The IMBs were
then washed with PBST and resuspended in an ABC-alkaline
phosphatase complex solution (Vector Laboratories, Inc.,
Burlingame, Calif.) for 30 min. The IMBs were washed three times
with PBST and resuspended in 100 .mu.l of 0.2 M Tris buffer
containing 0.1% BSA (pH 8.5) to remove unbound enzyme complex. A
40-.mu.l suspension of the IMBs was added to 3 ml of Fluorophos
substrate (Advanced Instruments, Norwood, Mass.) and fluorescence
monitored for 2 min at 38.degree. C. in a Fluorophos FLM200
fluorometer (Advanced Instruments, Norwood, Mass.).
[0100] As shown in FIG. 2, using immunocapture-sandwich ELISA,
spores in UHT skim milk were quantified down to 8.times.10.sup.3
cfu/ml in 2 h with no pre-enrichment steps and no sample
preparation. Increasing the number of beads in the assay increased
the fluorescence activity, suggesting that this could further
increase the assay sensitivity (P. M. Fratamico et al., supra; E.
Skjerve et al., supra).
[0101] The slope of the generated curves was similar for all
samples tested, indicating that sample background did not grossly
influence antigen binding (FIG. 3). Therefore, approximate spore
loads can be obtained without calibrating the assay to each
product. Foods containing fat, such as raw whole milk, required a
longer time for separation of the IMBs and gentle removal of
supernate to avoid trapping the beads in the fat and removing them
with the supernate. Separation of IMBs from fatty products required
5 min rather than the 2 min used for nonfat samples. Soil samples
containing a high percentage of iron fines interfered with bead
recovery, although other soil types tested did not. These data
support the use of this assay to test for B. stearothermophilus
spores in food and environmental sample.
[0102] Since the assay has been designed to be used with raw
ingredients that may vary in temperature, the ability of the IMBs
to capture B. stearothermophilus spores at temperatures ranging
from 4.degree. C. to 55.degree. C. was tested. IMBs were added to 1
ml UHT skim milk containing 5.times.10.sup.4 B. stearothermophilus
spores and incubated between 4.degree. C. to 55.degree. C. while
rotating (50 rpm) for 30 min. The IMB were washed four times with
PBST, plated on PCA, and incubated overnight at 65.degree. C. B.
stearothermophilus colonies were counted to quantitate bound
spores. Regardless of the temperature of the sample, the number of
spores captured from UHT skim milk containing 5.times.10.sup.4 B.
stearothermophilus spores did not vary significantly. This means
that sample preparation time can be reduced. These data suggest
that this approach is over 100 times more sensitive than the only
other rapid spore assay (Y. H. Chang & P. M. Foegeding, supra),
is about 10 times faster than any spore assay with equivalent
sensitivity (G. H. Richardson, supra), and can be used to
quantitate a single species of spore in a mixed spore population in
chemically complex backgrounds.
[0103] While detection of spores was achieved with immunomagnetic
beads, it was believed that sensitivity and efficiency could be
improved by using a fluidized bed capture system. Hence, the
capture step was fluidized by immobilizing antibodies onto larger
beads ranging is size from 1-7 mm. Use of a fluidized bed module
(FIG. 4) further increased the sensitivity of the assay to less
than a single cell per ml of liquid and allowed the assay to be
done without pre-incubation and to obtain a finished result within
30 min in all the samples tested (FIG. 5). FIG. 4 shows a schematic
representation of an illustrative module 30 comprising a housing 34
having an inlet opening 38 for flow of a liquid medium to be tested
into the module. A plate 46 having holes therein to permit flow of
the liquid through the module is placed with the plane of the plate
generally perpendicular to the direction of flow of the liquid. The
beads 50 are placed upstream of the plate. Arrow 54 shows the
direction of flow of liquid through the module. The size of the
holes in the plate is selected to be smaller than the size of the
beads such that the beads cannot pass through the outflow opening
42. In an illustrative embodiment of the module, the holes in the
plate were 3 mm in diameter, and the beads were 7 mm in diameter.
The housing and plate should be constructed of materials, such as
stainless steel and high durability plastics, having high
durability and compatibility with liquids of various types.
EXAMPLE 15
[0104] In this example, 0.1 M phosphate buffer, pH 7.2, containing
various concentrations of B. globigii spores was passed through an
immunoflow module containing 7 mm ceramic beads having anti-B.
globigii antibodies conjugated thereto according to the procedure
of Example 6. The buffer was pumped through the module at 2 L/min.
After capture of the spores, all of the beads were removed from the
module, and a solid phase ELISA using biotinylated anti-B. globigii
antibodies to amplify the signal was performed according to the
procedure of Blake & Weimer, supra. The signal was read at 410
nm in a Biospec 1601 (Shimadzu Scientific Corp., Columbia, Md.) and
compared to a standard curve. FIG. 5 shows that spores could easily
be detected at concentration of less than 1 cfu/ml.
[0105] In a companion experiment, the following foods were tested
for the presence of B. globigii spores by immunoflow capture: raw
whole milk, skim milk, raw hamburger, canned green beans, canned
corn, canned peas, canned carrots, canned mixed vegetables, canned
spinach, beer, fermented sausage, Vienna sausage, raw chicken,
canned chicken, canned pork and beans, canned kidney beans, fresh
sliced mushrooms, and canned tomato sauce. Fluid products were
tested without modification. Other products were dissolved or
suspended at 1 g/ml. Fifty ml of product was pumped through the
module at a rate of 2.5 L/min. Bound spores were quantitated using
fluorescence detection as described above. B. globigii spores were
detected in each of these foods at a concentration of 1
spore/ml.
[0106] This is a significant improvement over prior results and
provided a method for increasing the sample size that could be
used. Use of immunoflow at 2-4 L/min allowed detection in less time
and in the presence of fat or protein that interfered with
immunomagnetic detection and some foods. A characteristic dip at
10.sup.3 spores/ml was found, which is commonly observed. The cause
for this deviation is unknown but is linked to the lower flow rate
used, since this dip is not noticeable at flow rates>4L/min. The
dynamic range is at least nine decades, suggesting this module will
not easily be overloaded in the field.
EXAMPLE 16
[0107] In this example, the procedure of Example 15 was carried out
except that river water (pH 8.5) with added B. globigii spores
(10.sup.3 spores/ml) was pumped through the module at a flow rate
from 1 to 4 L/min for times ranging from 1 to 180 minutes.
Detection and spore capture increased as the flow rate increased,
with the maximum detection at 15 minutes and a flow rate of 4
L/min. Detection decreased as the flow rate decreased and as the
flow time increased. These data suggest a complex interaction
between the capture surface and the spore is occurring, but that it
is not matrix dependent. Similar results were observed with
detection of B. globigii spores in PBS and penicillin in milk at
114 L/min. Additionally, the results obtained with penicillin
detection suggests that the range of flow rates for capture and
detection is large, at least 1-114 L/min.
EXAMPLE 17
[0108] To demonstrate the use of immunoflow with small protein
targets, BSA detection was done using immunoflow with 5 mm glass
beads modified with dextran and anti-BSA antibodies and a flow rate
of 2 L/min. The assay detected BSA over a broad range (FIG. 6), and
had a lower detection limit of at least 0.075 ng/ml without the
avidin/biotin complex for amplification (FIG. 7). These data
confirm the use of immunoflow detection for use with chemicals
(penicillin), small proteins (BSA), and microbes (Bacillus spores)
at flow rates varying from 1-114 L/min.
[0109] FIG. 8 shows an illustrative embodiment of the present
invention for capturing and detecting an antigen from a sample.
This illustrative composition 100 comprises an antibody 104 coupled
to the surface of a solid support, for example, a bead 108. The
antibody 104 is coupled to the bead 108 by means of a spacer 112.
As described above, spacers can include dextran, polythreonine,
polyserine, polyethylene glycol, biotin/avidin linkages, and the
like. The spacer 112 is coupled to the surface of the bead 108 by a
chemical linker 116, and the antibody 104 is also coupled to the
spacer 112 by a chemical linker 120. A bacterium 124 captured by
the composition 100 is shown bound to the antibody 104.
[0110] FIG. 9 shows detection of the bacterium 124 captured as
illustrated in FIG. 8. Detection is by a high flow rate solid phase
ELISA according to methods well known in the art. A secondary
antibody 128 is linked to an enzyme 132 by means of a chemical
linker 136 for providing an amplification complex 140 for providing
a detectable signal when a suitable substrate is placed in contact
with the amplification complex 140. Illustrative substrates that
can be used in accordance with the present invention include those
that yield visual, calorimetric, luminescent, and fluorescent
signals upon digestion of the substrate by the enzyme.
[0111] Methods for detecting antibody/antigen or immune complexes
are well known in the art. The present invention can be modified by
one skilled in the art to accommodate the various detection methods
known in the art. The particular detection method chosen by one
skilled in the art depends on several factors, including the amount
of sample available, the type of sample, the stability of the
sample, the stability of the antigen, and the affinity between the
antibody and antigen.
[0112] While these techniques are well known in the art, examples
of a few of the detection methods that could be used to practice
the present invention are briefly described below.
[0113] There are many types of immunoassays known in the art. For
example, a common type of immunoassay is a non-competitive sandwich
or capture assay, such as enzyme-linked immunosorbent assays
(ELISA). In a non-competitive capture ELISA, unlabeled antigen is
captured by an antibody bound to a solid support, such as the
surface of the bead as illustrated in FIG. 8. After the immune
complexes have formed, excess sample is removed and the bead is
washed to remove nonspecifically bound antigen. If the
concentration of the antigen in the sample is sufficiently dilute,
however, it may not be necessary to remove nonspecifically bound
antigens because such antigens are present in such low amounts. The
immune complexes are then reacted with an appropriate
enzyme-labeled antibody (secondary antibody), which recognizes the
same or a different epitope on the antigen as the primary antibody.
The secondary antibody reacts with antigens in the immune
complexes. After a second wash step, the enzyme substrate is added.
The enzyme linked to the secondary antibody catalyzes a reaction
that converts the substrate into a product. Hence, enzyme activity
is directly proportional to the amount of antigen in the biological
sample. D. M. Kemeny & S. J. Challacombe, ELISA and Other Solid
Phase Immunoassays (1988). Illustratively, the product is
fluorescent or luminescent, which can be measured using technology
and equipment well known in the art. It is also possible to use
reaction schemes that result in a colored product, which can be
measured spectrophotometrically, but such calorimetric reactions
are not preferred.
[0114] Typical enzymes that can be linked to secondary antibodies
include horseradish peroxidase, glucose oxidase,
glucose-6-phosphate dehydrogenase, alkaline phosphatase,
.beta.-galactosidase, and urease. Secondary antigen-specific
antibodies linked to various enzymes are commercially available
from, for example, Sigma Chemical Co. and Amersham Life Sciences
(Arlington Heights, Ill.).
[0115] Fluorescence immunoassays can also be used when practicing
the method of the present invention. Fluorescence immunoassays are
similar to ELISAs except the enzyme is substituted for fluorescent
compounds called fluorophores or fluorochromes. These compounds
have the ability to absorb energy from incident light and emit the
energy as light of a longer wavelength and lower energy.
Fluorescein and rhodamine, usually in the form of isothiocyanates
that can be readily coupled to antigens and antibodies, are most
commonly used in the art. D. P. Stites et al., Basic and Clinical
Immunology (1994). Fluorescein absorbs light of 490 to 495 nm in
wavelength and emits light at 520 nm in wavelength.
Tetramethylrhodamine absorbs light of 550 nm in wavelength and
emits light of 580 nm in wavelength. Illustrative
fluorescence-based detection methods include ELF-97 alkaline
phosphatase substrate (Molecular Probes Inc., Eugene, Oreg.);
PBXL-1 and PBXL-3 (phycobilisomes conjugated to streptavidin)
(Martek Biosciences Corp., Columbia, Md.); FITC and Texas Red
labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories,
Inc., West Grove, Pa.); and B-Phycoerythrin and R-Phycoerythrin
conjugated to streptavidin (Molecular Probes Inc.). ELF-97 is a
nonfluorescent chemical that is digested by alkaline phosphatase to
form a fluorescent molecule. Because of turn over of the alkaline
phosphatase, use of the ELF-97 substrate results in signal
amplification. Fluorescent molecules attached to secondary
antibodies do not exhibit this amplification.
[0116] Phycobiliproteins isolated from algae, porphyrins, and
chlorophylls, which all fluoresce at about 600 nm, are also being
used in the art. I. Hemmila, Fluoroimmunoassays and
Immunofluorometric Assays, 31 Clin. Chem. 359 (1985); U.S. Pat. No.
4,542,104. Phycobiliproteins and derivatives thereof are
commercially available under the names R-phycoerythrin (PE) and
Quantum Red.TM. from, for example, Sigma Chemical Co.
[0117] In addition, Cy-conjugated secondary antibodies and antigens
are useful in immunoassays and are commercially available. Cy-3,
for example, is maximally excited at 554 nm and emits light of
between 568 and 574 nm. Cy-3 is more hydrophilic than other
fluorophores and thus has less of a tendency to bind
nonspecifically or aggregate. Cy-conjugated compounds are
commercially available from Amersham Life Sciences.
[0118] Illustrative luminescence-based detection methods include
CSPD and CDP star alkaline phosphatase substrates (Roche Molecular
Biochemicals); and SuperSignal.RTM. horseradish peroxidase
substrate (Pierce Chemical Co., Rockford, Ill.).
[0119] Chemiluminescence, electroluminescence, and
electrochemiluminescenc- e (ECL) detection methods are also
attractive means for quantifying antigens and antibodies in a
sample. Luminescent compounds have the ability to absorb energy,
which is released in the form of visible light upon excitation. In
chemiluminescence, the excitation source is a chemical reaction; in
electroluminescence the excitation source is an electric field; and
in ECL an electric field induces a luminescent chemical
reaction.
[0120] Molecules used with ECL detection methods generally comprise
an organic ligand and a transition metal. The organic ligand forms
a chelate with one or more transition metal atoms forming an
organometallic complex. Various organometallic and transition
metal-organic ligand complexes have been used as ECL labels for
detecting and quantifying analytes in biological samples. Due to
their thermal, chemical, and photochemical stability, their intense
emissions and long emission lifetimes, ruthenium, osmium, rhenium,
iridium, and rhodium transition metals are favored in the art. The
types of organic ligands are numerous and include anthracene and
polypyridyl molecules and heterocyclic organic compounds. For
example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl, and
derivatives thereof, are common organic ligands in the art. A
common organometallic complex used in the art includes
tris-bipyridine ruthenium (II), commercially available from IGEN,
Inc. (Rockville, Md.) and Sigma Chemical Co.
[0121] Advantageously, ECL can be performed under aqueous
conditions and under physiological pH, thus minimizing biological
sample handling. J. K. Leland et al., Electrogenerated
Chemiluminescence: An Oxidative-Reduction Type ECL Reactions
Sequence Using Triprophyl Amine, 137 J. Electrochemical Soc.
3127-3131 (1990); WO 90/05296U.S. Pat. No. 5,541,113. Moreover, the
luminescence of these compounds may be enhanced by the addition of
various cofactors, such as amines.
[0122] In practice, a tris-bipyridine ruthenium (II) complex, for
example, may be attached to a secondary antibody using strategies
well known in the art, including attachment to lysine amino groups,
cysteine sulfhydryl groups, and histidine imidazole groups. After
washing nonspecific binding complexes, the tris-bipyridine
ruthenium (II) complex would be excited by chemical, photochemical,
and electrochemical excitation means, such as by applying current
to the bead. E.g., WO 86/02734. The excitation would result in a
double oxidation reaction of the tris-bipyridine ruthenium (II)
complex, resulting in luminescence that could be detected by, for
example, a photomultiplier tube. Instruments for detecting
luminescence are well known in the art and are commercially
available, for example, from IGEN, Inc.
[0123] FIGS. 10 and 11 show a module 150 or bead holder into which
the bead-bound antibodies are placed. The module comprises a
transparent plastic tube body 154 that holds the beads 158 in a
column format. The tube body comprises a top end 162 and a bottom
end 166. A top cap 170 is disposed on the top end of the tube body,
and a bottom cap 174 is disposed on the bottom end of the tube
body. The bottom cap 174 comprises an inlet 178 for permitting
liquid to enter the module, and the top cap 170 comprises an outlet
182 for permitting liquid to exit the module. The outlet 182 and
the inlet 178 are configured for being received in the lumen of
tubing for linking the module to liquid handling systems, as will
be described in more detail momentarily. A porous screen 186 is
disposed in the cavity 190 formed by the tube body and the top cap
and the bottom cap adjacent to both the top end 162 and the bottom
end 166 of the tube body. These porous screens 186 are configured
to permit liquid to pass therethrough while retaining the beads 158
in the cavity 190.
[0124] Once the antibodies have been attached to beads as described
herein, the beads are loaded into the module. Typically, 10 to 280
beads are used. Illustratively, 55 beads (2 g of 3 mm diameter
beads) may be used.
[0125] FIG. 12 shows the module 200, as described above, connected
to components of a system for use in detecting an antigen in a
sample. The sample 204 is contained in a vessel 208. A section of
tubing 212 is coupled to the inlet 216 of the module 200, and the
distal end 220 of the tubing 212 is placed in the sample 204.
Another section of tubing 224 is coupled to the outlet 228 of the
module 200, and the distal end 232 of the tubing 224 is coupled to
a vacuum pump 236. A photomultiplier tube 240 or PMT is mounted
adjacent to the module 200 such that when the beads are at rest,
the PMT 240 is above the level of the beads 244. The PMT 240 is
coupled to signal detection electronics 248 for detecting and
counting signals produced by the PMT 240.
[0126] The apparatus of FIG. 12 is used by placing a sample 204 in
the vessel 208, and then causing the vacuum pump 236 to draw a
vacuum through the sections of tubing 224 and 212 and the module
200, thereby drawing the sample liquid 204 through tubing 212,
through the module 200, and through tubing 224. The sample can be
recirculated through the module through another section of tubing
252 or can be collected in a trap 256. A valve 260 in tubing line
252 controls whether the sample is recirculated or trapped. The
flow of the sample through the beads 244 causes the beads to be
fluidized in the cavity of the module. A portion of the antigens
contained in the sample are bound by bead-bound antibodies in the
module. After capture of the antigens, the beads can be washed by
placing the distal end 220 of tubing 212 in another vessel
containing a wash solution. Operation of the vacuum pump draws the
wash solution through the beads for washing away debris. After
washing of the beads, enzyme-labeled secondary antibodies are drawn
through the module in a similar manner. The enzyme-labeled
secondary antibodies bind to the captured antigens. Another washing
step can then be carried out. Next, a solution containing a
substrate for the enzyme is passed through the module such that the
substrate comes in contact with the enzyme. The substrate is
digested by the enzyme to result in a detectable signal, such as a
luminescent signal as described above. At this point, the vacuum
pump is turned off such that the beads in the module settle by
gravity to the bottom of the module. Once this has occurred, the
PMT is activated for detecting the signal, such as a luminescent
signal. Detection of the luminescent signal by the PMT results in
the production of an electronic signal, which is transmitted to the
signal detection electronics apparatus for detection, counting, and
analysis of the electronic signals. The number of electronic
signals detected and counted by the signal detection electronics is
proportional to the number of antigens captured in the module.
[0127] Illustratively, the vacuum pump can be set to draw a vacuum
of 2 to 5 inches of mercury. It has been determined through
experience that a setting of 5 inches of mercury is satisfactory
for capturing a target antigen on the surface of beads. This amount
of vacuum fluidizes the bead bed, and permits capture of sufficient
antigen for detection in about 5 minutes with 50 ml samples. FIG.
13 shows capture of E. coli O157:H7 from buffer on the bead surface
with fluorescently labeled cells. Each flow rate has an optimum
cycling time for achieving capture. A flow rate of about 150 ml/min
is convenient for use with a 50 ml sample. The data of FIG. 13 were
verified by doing plate counts, according to methods well known in
the art, in various foods in the same experimental design, but
looking at the remaining fluid in the tube at the maximum capture
settings for 150 ml/min (FIG. 14). In each food type, the fluidized
bed of beads with anti-E. coli O157:H7 attached thereto captured
the cells during the sampling time at 150 ml/minute. In each sample
type, the cells were specifically captured onto the bead surface.
Comparison of FIGS. 13 and 14 indicates that the beads capture the
cells at approximately the same efficiency in both methods.
Therefore, the cells were specifically captured by the
antibody-modified beads.
[0128] Once the target antigen is captured and the debris is washed
away, the other reagents are added in the same manner, i.e., they
are caused to flow into the module with the vacuum of the same
setting. The antibodies bind the antigens, and any excess is washed
away from the beads.
[0129] Next, the substrate, such as Lumigen APS-5, is added and
then held in the module as the signal develops due to the
amplification complex (FIG. 15). This may be done with many types
of molecules, but horseradish peroxidase and alkaline phosphatase
are especially convenient. The signal is generated and is stable
within about 3 minutes. The useful portions of the curve are shown
in the circles (FIG. 15). The analysis can be done with a running
slope or an endpoint. The information needed to calculate the
running slope is available within the first 2 seconds of the
curve.
[0130] Once this curve is generated, it is used to determine the
amount of bacteria captured onto the bead surface (FIG. 16).
Comparison of the initial slopes or the endpoint allow the final
result to be determined. Based on these calculations, the cell
density in the original sample is determined and reported. Use of
the initial slopes is more discriminating, but the endpoint is
adequate for rough estimation of the mid-range concentrations.
[0131] The signal is detected with a photon-counting PMT specific
for luminescence, for example a Hamamatsu model H7360.
[0132] Table 3 shows the results of detection of E. coli O157:H7 by
the procedure of the present invention as compared to the results
achieved with known commercial tests. In all cases, the present
invention, termed ImmunoFlow, detected the added organism in each
food within 30 minutes without the need for culturing the organism.
The ImmunoFlow tests were done without pre-enrichment, whereas the
immunoprecipitation and lateral flow ELISA and all experiments with
bean sprouts were done with 24 hr of pre-enrichment.
3TABLE 3 Immunopreci- Lateral Food type ImmunoFlow BAM pitation
Flow ELISA Apple juice - - - - control Apple juice + + + + Beer - -
- - control Beer + + + + Hamburger - - - - control Hamburger + + +
+ Bean + + + + sprouts
EXAMPLE 18
[0133] In a 1-liter flask, 94 ml of double-distilled water and 1.88
g of dextran (Sigma, St. Louis, Mo.) were combined and mixed by
swirling the flask until all of the dextran was solubilized. The
flask was then wrapped in aluminum foil and 3.12 g of NaIO.sub.4
was added. The flask was then capped with foil and placed on a
shaker at low speed for 1 hour. Next, the flask was removed from
the shaker and 125 g of APTES glass beads (3 mm diameter) was
added. The flask was then returned to the shaker for 1 hour of
agitation at low speed. Following agitation, the beads were removed
from the flask and washed with 1.25 liter of double-distilled water
and then 125 ml of 50 mM sodium phosphate, pH 7.2. After washing,
the beads were returned to the original flask, which was rinsed
prior to the beads being replaced, and then 125 ml of 4 mM ADH, 50
mM sodium phosphate (pH 7.2). The pH of the buffer was then checked
to ensure that the pH was about neutral, and then the flask was
returned to the shaker for low speed agitation for 2 hours.
[0134] While the beads were shaking, the primary antibody solution
was prepared. The primary antibody (0-2 mg), 5 mg of NaIO.sub.4,
and enough PBS to raise the volume to 1.0 ml were combined in a 1.5
ml plastic tube (Eppendorf). The contents were mixed by vortexing
until all the ingredients had dissolved. Next, a 10-ml desalting
column was prepared by washing with 50 ml of PBS (pH 7.2). The
washed column was then loaded with the 1.0 ml solution of primary
antibody, followed by elution with 8 ml of PBS (pH 7.2). The eluate
was collected in a reclosable 15-ml plastic tube, and the presence
of the antibody in the eluate was confirmed by checking the
absorbance at 280 nm.
[0135] The flask containing the beads was then removed from the
shaker, and 125 mg of NaBH.sub.4 was added to the flask. The flask
was then returned to the shaker for 30 minutes at low speed. The
beads were then removed from the flask and washed successively with
1.25 liter of double-distilled water, 250 ml of 50 mM sodium
phosphate (pH 7.2) containing 1 M NaCl, and 250 ml PBS (pH 7.2).
The washed beads were then placed in a fresh 1-liter flask, to
which was added 113 ml of PBS (pH 7.2) and the primary antibody
solution. The flask was then placed on a shaker and the contents
were agitated at low speed for 1 hour. The foil-capped flask was
then chilled overnight at 4.degree. C.
[0136] The next day, 125 mg of NaBH.sub.4 was added to the flask,
and the flask was placed on a shaker and agitated at low speed for
30 minutes.
[0137] Following agitation, the beads were washed successively with
1.25 liter of 50 mM sodium phosphate (pH 7.2), 250 ml of 50 mM
sodium phosphate (pH 7.2) containing 1 M NaCl, and 250 mL Tris-HCl
(pH 7.2). After washing, the beads were poured into a flat glass
tray and covered with filter-sterilized 2% BSA/0.02% NaN.sub.3. The
beads were then separated into aliquots in sterile plastic
containers and covered with excess 2% BSA/0.02% NaN.sub.3. The
containers were then shaken at low speed for 1 hour with swirling
every 30 minutes. The beads were then stored at 4.degree. C. until
use.
EXAMPLE 19
[0138] Materials and Methods
[0139] Chemical Reagents
[0140] Borosilicate glass beads (3 mm diameter, 7.5.times.10.sup.-4
m.sup.2/g) were obtained from VWR Scientific Products.
3-Aminopropyl-triethoxysilane (APTES), succinic anhydride,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),
morpholineethanesulfonic acid (MES) ovalbumin, (OVA), and bovine
serum albumin (BSA) were obtained from Sigma Chemical Co. (St.
Louis, Mo.). PEG-dicarboxymethyl (MW 3,400) was obtained from
Shearwater Polymers, Inc. (Huntsville, Ala.) and the BCA protein
assay kit was obtained from Pierce Chemical, Co. (Rockford, Ill.).
Bacillus globigii (BG) spores were provided by Dugway Proving
Ground (Dugway, Utah). E.coli 0157:H7 was purchased from ATCC, and
rehydrated in Tryptic Soy Broth (TSB) at 37.degree. C. All other
reagents required in the coupling and wash buffers were analytical
grade.
[0141] Antibodies
[0142] Monoclonal mouse anti-chicken egg ovalbumin (anti-OVA, clone
OVA-14) and monoclonal mouse anti-BSA (anti-BSA, clone BSA-33) were
purchased from Sigma Chemical Co (St. Louis, Mo.). Polyclonal goat
anti-E.coil 0157:H7 was obtained from Kirkegaard & Perry
Laboratory (Gaithersburg, M.) Monoclonal goat anti-Bacillus
globigii was kindly provided by Dugway Proving Grounds (Dugway,
Utah).
[0143] Immobilization
[0144] Glass beads, 200 g, were cleaned in concentrated nitric acid
for 1 hr in a boiling water bath. Beads were derivatized with
3-aminopropyltriethoxy silane according to M. K. Walsh & H. E.
Swaisgood, Characterization of a chemically conjugated
beta-galactosidase bioreactor, 17 J. Food Biochem. 283-292 (1993).
Half of the beads, 100 g, were succinylated with succinic anhydride
in 0.1 M sodium acetate buffer, pH 4.0, for 2 hours. Dry succinic
anhydride, 10 g, was added to 150 ml of sodium acetate buffer for
succinylation. The APTES and succinylated glass beads were dried
overnight at 80.degree. C. and stored at room temperature.
[0145] Dicarboxymethyl-PEG was covalently attached to
APTES-modified glass beads using a one-step EDC reaction according
to G. T. Hermanson et al., Immobilized Affinity Ligand Techniques
80-83 (Academic Press, New York 1992). To 100 g beads, 100 ml of
0.1 M MES, (pH 4.5) containing 10 mM dicarboxymethyl-PEG and 500 mg
of EDC were added and incubated at 25.degree. C. with shaking (150
rpm) for 2 h. The PEG-modified beads were washed with PBS, pH 7.4,
and dried at 25.degree. C. Anti-OVA IgG, anti-BG IgG and anti-E.
coli IgG were attached to PEG and succinylated beads using the
one-step EDC reaction. To 100 g of beads, 1 mg of antibody in 150
mL of 0.1 M 0.1 M MES buffer (pH 4.5) was added. EDC, 500 mg, was
added and incubated at 25.degree. C. for 2 hours. After washing
antibody-modified beads (Ab-beads) 5 times with 50 ml PBST, BSA, 3%
in PBST, was added and incubated overnight to block nonspecific
binding sites on the glass surface.
[0146] The BCA protein assay was employed to determine the amount
of protein immobilized on the glass beads according to M. Bonde et
al., Direct dye binding-a quantitative assay for solid-phase
immobilized protein, 200 Anal. Biochem. 195-198 (1992), prior to
blocking with BSA. Antibody-modified beads, 8 g, were incubated
with 5 mL of BCA reagent for 30 min at 37.degree. C. The amount of
immobilized antibody was determined based on BSA as the
standard.
[0147] Detection of Captured OVA, BG Spores and E. coli 0157:H7
[0148] Capture of OVA, BG spores and E. coli 0157:H7 onto Ab-beads
was detected using a surface ELISA method. To 8 g of Ab-beads, 10
mL of appropriate antigen dilution (OVA, BG, or E. coli 0157:H7)
was added and incubated at 25.degree. C. for 1 h on a shaker (150
rpm). Beads were then washed five times with 50 mL PBST (pH 7.2).
Specific antibody (40 .mu.g/10 mL PBST) was added to the washed
beads and incubated at 25.degree. C. for 1 h at 150 rpm. Beads were
washed five times with 50 mL PBST (pH 7.2) before addition of
tertiary antibody, anti-IgG-HRP, 1 .mu.g in PBST. Beads were
incubated at 25.degree. C. for 1 h at 150 rpm followed by washing
five times with 50 mL PBST (pH 7.2). The substrate for HRP (5 mL of
tetramethyl benzidine) was added to the beads and incubated in the
dark for 15 min. The liquid, 1 ml, was removed from the beads and
the absorbance at 370 nm was measured with a Cary-100-Bio
Spectrophotometer (Varian Inst., Sugarland, Tex.).
[0149] Results and Discussion
[0150] Immobilized Antibodies
[0151] The results determined by the BCA protein assay indicated
that approximately the same amount of protein was immobilized onto
both the succinylated and PEG-modified beads. Considering the
surface area of the 3 mm glass beads (7.5.times.10.sup.-4
m.sup.2/g), the theoretical maximum amount of immobilized antibody
was 15 mg antibody/m.sup.2. These results are consistent with other
investigators, P. J. Soltys & M. R. Etzel, Equilibrium
Absorption of LDL and gold Immunoconjugates to Affinity Membranes
Containing PEG Spacers, 21 Biomaterials 37-48 (2000), for a
monolayer of immobilized antibody.
[0152] Comparison of Relative Capture Efficiency
[0153] The calibration plots for capturing OVA, BG spores and
E.coli 0157:H7 cells are shown in FIGS. 17A, B, and C. Signal at
370 nm indicates the amount of tertiary antibody, anti-IgG-HRP,
bound to the surface. Succinylated Ab-beads captured OVA, BG spores
and E. coli 0157:H7, but the capture efficiency was less than the
PEG Ab-beads. The slope of the PEG Ab-beads are higher compared to
the succinylated Ab-beads for each antigen tested. The influence of
a PEG spacer is more dramatic in the capture of E. coli
O157:H7.
[0154] The observed difference in capture efficiency of PEG versus
succinylated Ab-beads can be explained by the long arm PEG provides
which distances the antibodies from the support surface. This
allows greater accessibility of the antigens to the immobilized
antibodies, reducing the amount of steric hindrance. Since the
total amount of antibodies immobilized onto succinylated and PEG
beads was similar, the antibodies immobilized via a spacer may have
been able to capture the antigen more effectively.
EXAMPLE 20
[0155] Surface Modification. The capture ability of antibodies
attached to different spacers was investigated (see Table 4).
Dextran (MW 37,500; Sigma, St. Louis, Mo.), polyethylene
glycol-dicarboxylmethyl (PEG, MW 3,400; Shearwater Polymers, Inc.,
Huntsville, Ala.), or polythreonine (MW 12,100; Sigma, St. Louis,
Mo.) were used as spacers.
[0156] Anti-BSA Ab were bound to 2.8 .mu.m tosyl-activated
polystyrene Dynalbeads (1 mg). Polythreonine (MW 12,100; Sigma) was
used as spacer and attached to the beads by the method of M. Blake
& B. C. Weimer, Immunomagnetic detection of Bacillus
stearothermophilus spores in food and environmental samples, 63
Appl. Environ. Microbiol. 1643-1646 (1997). A total of 100 .mu.l
(10.sup.8 total beads) modified polystyrene beads were used for
each sample. Anti-OVA Ab at a concentration of 10.sup.16
molecules/m.sup.2 were bound to 3 mm glass beads by the method as
described above. PEG was used as spacer and attached using the EDC
facilitated reaction. G. T. Hermanson, supra. Anti-B. globigii
spore Ab were bound to 3 mm glass and 7 mm ceramic beads.
Polythreonine was used as the spacer for the ceramic beads, whereas
PEG and dextran were used as spacers with glass beads. G. T.
Hermanson, supra. The antibody concentration was 10.sup.16
molecules/m.sup.2 for all anti-B. globigii spore beads. Anti-E.
coli O157:H7 (Kirkegaard & Perry Laboratory, Gaithersburg, Md.)
was attached to 3 mm glass beads using PEG as the spacer (as
described above) at a concentration of 10.sup.13 molecules/m.sup.2.
Hybridization slides (2.4 cm.sup.2 surface area) were also modified
with the same concentration of anti-E. coli O157:H7 antibodies
using PEG as the spacer.
[0157] Detection in Static Environment. Eight grams of Ab modified
beads were placed into a 50 ml centrifuge tube and 10 ml of sample
was added to the beads. Samples were incubated on a rocker for 1 h
at 25.degree. C. The samples were washed six times each with 50 ml
PBST (pH 5.8). Secondary Ab was added (total of 10.sup.12 molecules
of anti-E. coli O157:H7, 10.sup.13 molecules of anti-OVA, 10.sup.13
molecules of anti-BSA, and 10.sup.12 of anti-Bacillus globigii) in
10 ml PBST and beads were again incubated for 1 h. Samples were
washed six times with 50 ml PBST (pH 5.8) and incubated with 10 ml
of anti-IgG conjugated to horseradish peroxidase (Pierce Chemical
Company, Rockford, Ill.; IgG-HRP, 1 .mu.g/10 ml PBST, pH 5.8).
After the last wash step, beads were added to 5 ml of 1-Step Turbo
TMB-ELISA substrate (Pierce) and incubated in the dark for 20 min
before a reading was taken at A.sub.370 using a Cary 100-Bio
UV/Visible spectrophotometer (Varian, Sugar Land, Tex.). Water
blanks were used to zero the instrument.
[0158] Detection using Flow. Flow used a fluidized bed of beads, 8
g for the small unit and 250 g for the large unit, with Ab
covalently bound. To generate flow, a vacuum pump was used. The
reagents were evacuated from the bead cartridge through the top of
the reactor at a constant rate of 0.4 L/min (or 5" of Hg). As soon
as all the liquid passed over the beads the next reagent was
allowed to flow through the reactor. This continued until all the
reagents flowed across the beads. Just before adding the substrate
(TMB) to the bead cartridge, the vacuum was turned off and the TMB
was pulled into the reactor with a syringe. Once the TMB solution
covered the beads, the cartridge was sealed and placed in the dark
for 20 min. To measure the color development at A.sub.370, 1 ml of
the substrate was placed in a cuvette. Water blanks were used to
zero the spectrophotometer.
[0159] Four liters of 0.25 M sodium phosphate buffer (pH 7.0), or
river water were spiked with 10.sup.6 total Bacillus globigii
spores. A stainless steel module was filled with 250 g modified
anti-B. globigii spore ceramic beads. The B. globigii spore
solution was recycled over the 7 mm modified ceramic beads for 60
min at 1, 2, and 4 L/min flow rates. Five beads were taken out
every 15 min, replaced by 5 non-modified ceramic beads, and capture
ability of the beads investigated using the static method. At the
same time spore counts were determined on plate count agar.
[0160] The ability of the detection system to recover B. globigii
spores from various environmental and industrial water samples was
also investigated. Samples were collected from various
environmental and industrial locations in Cache Valley, Utah: (A)
Logan River water (pH 8.4); (B) Gossner's Cheese Plant tank water
(pH 9.2); (C) PBST (pH 7.2); and (D) Utah State University Dairy
Plant slush tank (pH 7.2). Samples were tested in flow using 8 g of
Ab modified beads. Standard curves were generated in these samples
with pure cultures in buffer. The ability of the detection system
to recover E. coli O157:H7 from meat extract and PBST samples was
also investigated with 10.sup.4 total cells and anti-E. coli
O157:H7 Ab attached to 3 mm glass beads via PEG.
4TABLE 4 List of antibodies and their modifications used to capture
bovine serum albumin (BSA), egg albumin (OVA), B. globigii spores,
and E. coli O157:H7. Antibody Bead Size Spacer Matrices tested
Anti-BSA polystyrene 2.8 .mu.m polythreonine PBS Anti-OVA Glass 3
mm PEG PBS Anti- Glass 3 mm PEG and Environmental B. globigii
dextran and industrial spores Ceramic 7 mm polythreonine water
samples, 0.25 M sodium phosphate buffer, pH 7.2 Anti-E. coli Glass
3 mm PEG PBS, meat O157:H7
[0161] Results
[0162] Static capture ability of modified beads. FIG. 18 shows the
standard curve obtained with anti-BSA-modified immunomagnetic beads
and static detection. Ab modified polystyrene beads, 10.sup.8 total
beads, successfully captured BSA. Very small amounts (<1 ng) of
BSA can be detected with these beads. The linear response of signal
to BSA increase was 99.7%, which makes this test very
sensitive.
[0163] FIG. 19 shows the standard curve obtained for 3 mm
PEG-anti-OVA-modified beads tested in static. The lower limit of
detection is 0.2 .mu.g. There is a linear response of signal
increase to OVA increase between 0.2 to 4.0 .mu.g. We did not test
beyond 4 .mu.g, because our objective was to develop a test that
was sensitive on the lower end.
[0164] Flow Capture Ability of Modified Beads.
[0165]
[0166] FIG. 20A shows the standard curves obtained for B. globigii
spore capture at a constant sample flow of 0.4 m L/min using 8 g of
3 mm PEG modified glass beads. A linear increase in signal was
observed as spore concentration increased. FIG. 20B shows the
Immuno capture of E. coli O157:H7 at a constant sample flow rate of
0.4 L/min with 8 g of 3 mm PEG modified glass beads. To observe the
influence of sample composition, PBST and sterile meat extract were
used. Meat extract spiked with cells consistently showed a higher
signal as compared to cells spiked into buffer.
[0167] FIG. 21 shows the standard curve obtained for B. globigii
spores spiked into the various environmental and industrial water
samples. All samples were sterilized prior to the addition of B.
globigii spores. No linear response to increased spore
concentration was observed using river or slush tank water. This
results in a constant flat line as seen in FIG. 21. A linear
response was observed using tank water from Gossner's Cheese Plant
and PBST. The lower limit of detection for both tank water and PBST
was 1 spore/sample. However, the upper limit with PBST was 10.sup.5
total spores and for tank water 10.sup.3 total spores. All
environmental and industrial samples had pHs values ranging from
7.2 to 9.2. The beads were active and captured spores over this
range.
* * * * *